F-box proteins and cancer: an update from functional and regulatory mechanism to therapeutic clinical prospects.

E3 ubiquitin ligases play a critical role in cellular mechanisms and cancer progression. F-box protein is the core component of the SKP1-cullin 1-F-box (SCF)-type E3 ubiquitin ligase and directly binds to substrates by various specific domains. According to the specific domains, F-box proteins are further classified into three sub-families: 1) F-box with leucine rich amino acid repeats (FBXL); 2) F-box with WD 40 amino acid repeats (FBXW); 3) F-box only with uncharacterized domains (FBXO). Here, we summarize the substrates of F-box proteins, discuss the important molecular mechanism and emerging role of F-box proteins especially from the perspective of cancer development and progression. These findings will shed new light on malignant tumor progression mechanisms, and suggest the potential role of F-box proteins as cancer biomarkers and therapeutic targets for future cancer treatment.

An integrative pan-cancer analysis of biological and clinical impacts underlying ubiquitin-specific-processing proteases.

Ubiquitin-specific-processing proteases (USPs), the largest deubiquitinating enzyme (DUB) subfamily, play critical roles in cancer. However, clinical utility of USPs is hindered by limited knowledge about their varied and substrate-dependent actions. Here, we performed a comprehensive investigation on pan-cancer impacts of USPs by integrating multi-omics data and annotated data resources, especially a deubiquitination network. Meaningful insights into the roles of 54 USPs in 29 types of cancers were generated. Although rare mutations were observed, a majority of USPs exhibited significant expressional alterations, prognostic impacts and strong correlations with cancer hallmark pathways. Notably, from our DUB-substrate interaction prediction model, additional USP-substrate interactions (USIs) were recognized to complement knowledge gap about cancer-relevant USIs. Intriguingly, expression signatures of the USIs revealed clinically meaningful cancer subtypes, where key USPs and substrates cooperatively contributed to significant prognosis differences among subtypes. Overall, this investigation provides a valuable resource to assist mechanism research and clinical utility about USPs.

A Multidimensional Characterization of E3 Ubiquitin Ligase and Substrate Interaction Network.

E3 ubiquitin ligases (E3s) play a critical role in molecular and cellular mechanisms. However, a large number of E3-substrate interactions (ESIs) remain unrevealed. Here, we integrated the increasing omics data with biological knowledge to characterize and identify ESIs. Multidimensional features were computed to obtain the association patterns of ESIs, and an ensemble prediction model was constructed to identify ESIs. Comparison with non-ESI cases revealed the specific association patterns of ESIs, which provided meaningful insights into ESI interpretation. Reliability of the prediction model was confirmed from various perspectives. Notably, our evaluations on leucine-rich repeat family of F box (FBXL) family were consistent with a proteomic study, and several substrates for SKP2 and an orphan E3 FBXL6 were experimentally verified. Moreover, a cancer hallmark ESI landscape was studied. Taken together, our study catches a glimpse at the omics-driven ESI association patterns and provides a valuable resource (http://www.esinet.dicp.ac.cn/home.php) to assist ubiquitination research.

Metabolomics profiling of metformin-mediated metabolic reprogramming bypassing AMPKα.

BACKGROUND: Metformin is a first-line drug for treating type 2 diabetes and has gained considerable interest as a potential anticancer agent. Increasing evidence suggests that metformin antagonizes diabetes and tumors through disrupting metabolic homeostasis and altering energy state. However, whether AMP activated protein kinase (AMPK) contributes to such effects of metformin remains controversial. METHODS: We performed integrative metabolomics analyses to systematically examine the effects of metformin on metabolic pathways in Prkaa1 wild type (WT) and knock-out (KO) mouse embryonic fibroblast (MEF) cells as well as human cells based on gas chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry (CE-MS). RESULTS: Metformin treatment induced metabolic reprogramming and reduced the energy state of both Prkaa1 WT and KO MEF cells, as evidenced by suppressed tricarboxylic acid (TCA) cycle, elevated lactate production as well as decreased NAD+/NADH ratio. Additionally, metabolic flux analysis also showed that metformin Ampkα-independently increased metabolic flux from glucose to lactate and decreased metabolic flux from acetyl-CoA to TCA cycle as well as from pyruvate to malate. Moreover, metformin Ampkα-dependently upregulated P-Acc but Ampkα-independently inhibited the levels of P-mTor, P-S6, Lc3, Atgl and P-Erk in MEF cells. Similarly, we demonstrated that a commonly used AMPK agonist 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) and fetal bovine serum (FBS) starvation, as a common model for energy stress, both led to Ampkα-independent metabolism alterations in MEF cells. Furthermore, these effects of metformin were also confirmed in human hepatocellular carcinoma (HCC) cells as well as in MCF10A shControl and shPRKAA1 cells. Importantly, we found that metformin could obviously inhibit colony conformation of HCC cells in an Ampkα-independent manner. CONCLUSIONS: Our data highlight a comprehensive view of metabolic reprogramming mediated by metformin as well as AICAR. These observations suggest that metformin could affect cellular metabolism largely bypassing Ampkα, and may provide a new insight for its clinical usage.

USP10 suppresses tumor progression by inhibiting mTOR activation in hepatocellular carcinoma.

Dysregulation of deubiquitination pathway is associated with poor prognosis in cancers such as hepatocellular carcinoma (HCC). The mammalian target of rapamycin, mTOR, has become an attractive cancer therapeutic target in HCC. However, whether and how aberrant expression of deubiquitination pathway regulates mTOR pathway has remained elusive. Here we report that ubiquitin-specific protease 10 (USP10) functions as a tumor suppressor which inhibits mTOR pathway by stabilizing PTEN and AMPKα in HCC cells. Mechanistically, USP10 interacts and stabilizes PTEN and AMPKα by inhibiting their polyubiquitylation. This stabilization in turn inhibits AKT phosphorylation and mTOR Complex1 (mTORC1) activation. In human liver cancer, USP10 expression is downregulated in HCC tumor tissues across three independent HCC cohorts, and lower-expression of USP10 will generate poor prognosis outcome. Collectively, our results uncover an undescribed mechanism where USP10, as a tumor suppressor, negatively regulates mTORC1 activation and AKT phosphorylation by stabilizing AMPKα and PTEN in HCC cells. This study sheds light on the theoretical basis of mTOR signaling pathway-oriented targeting treatment in clinic.

Epigenetic Downregulation of PTEN in Gallbladder Cancer.

PURPOSE: Gallbladder cancer (GBC) is becoming a global health problem. Phosphatase and tensin homolog (PTEN), also known as guardian gene of cancer, encodes a protein, which dephosphorylates phosphatidylinositol (3,4,5) triphosphate {PtdIns (3,4,5) P3 } into phosphatidylinositol (4,5) diphosphate {PtdIns (4,5) P2} that results in the inhibition of AKT/PKB signaling pathway. PTEN plays a key role in the pathogenesis of various cancers. However, its role in GBC is still obscure. The present study was aimed to identify the epigenetic role of PTEN in GBC and GSD. METHODS: PTEN promoter methylation was studied by methylation-specific PCR in 50 GBC and 30 GSD tissues. Transcript level expression of PTEN was analyzed by reverse transcriptase PCR and quantitative PCR in 20 GBC and 20 GSD tissue samples. Immunohistochemistry was performed on tissue microarrays of 136 GBC samples to correlate the methylation pattern with the pattern of in situ expression of PTEN protein. Student's t test was performed to test the significant level of difference between case and control samples. Values showing p ≤ 0.05 were considered significant. RESULTS: MS PCR showed PTEN promoter methylation in 30% (15/50) GBC (p = 0.0486) and 22.86% (8/30) GSD (p = 0.0530) cases. RT-PCR and qPCR revealed downregulation of PTEN in advanced GBC cases (p < 0.0001), but, not in GSD (p = 0.901). Immunohistochemistry of GBC tissue microarray scored 1+ in 20.29% (p = 0.0028) and zero or negative in 50% (p < 0.0001) GBC cores. CONCLUSIONS: Thus, PTEN may be considered as a useful biomarker for the management of GBC, however, needs larger sample size to validate it further.

Epigenetic regulation of APC in the molecular pathogenesis of gallbladder cancer.

BACKGROUND & OBJECTIVES: Loss of function of adenomatous polyposis coli (APC) has been reported in cancer. The two promoters of APC, 1A and 1B also have roles in cancer. But, the epigenetic role of APC promoters is not yet clear in gallbladder cancer (GBC) and gallstone diseases (GSD). We undertook this study to determine the epigenetic role of APC in GBC and GSD. METHODS: Methylation-specific (MS)-PCR was used to analyze the methylation of APC gene. The expression of APC gene was studied by semi-quantitative PCR, real-time PCR and immunohistochemistry (IHC) in GBC, GSD and adjacent normal tissues. RESULTS: Of the two promoters, APC 1A promoter was found methylated in 96 per cent GBC ( P=0.0155) and 80 per cent GSD (P=0.015). Exon 1 was downregulated in grade II (P=0.002) and grade III (P=0.0001) of GBC, while exon 2 was normally expressed. Scoring analysis of IHC revealed 0 or negativity in 34.48 per cent (P=0.057) and 1+ in 24.14 per cent (P=0.005) GBC cases suggesting loss of APC expression. INTERPRETATION & CONCLUSIONS: The present findings indicate epigenetic silencing of APC in advanced GBC. The methylation pattern, followed by expression analysis of APC may be suggested for diagnostic, prognostic and therapeutic purposes in GBC in future.

Epigenetic regulation in gallbladder cancer: Promoter methylation profiling as emergent novel biomarkers.

DNA methylation, once considered to rule the sex determination in Mary Lyon's hypothesis, has now reached the epicenter of human diseases, from monogenic (e.g. Prader Willi syndrome, Angelman syndromes and Beckwith-Wiedemann syndrome) to polygenic diseases, like cancer. Technological developments from gold standard to high throughput technologies have made tremendous advancement to define the epigenetic mechanism of cancer. Gallbladder cancer (GBC) is a fatal health issue affecting mostly the middle-aged women, whose survival rate is very low due to late symptomatic diagnosis. DNA methylation has become one of the key molecular mechanisms in the tumorigenesis of gallbladder. Various molecules have been reported to be epigenetically altered in GBC. In this review, we have discussed the classes of epigenetics, an overview of DNA methylation, technological approaches for its study, profile of methylated genes, their likely roles in GBC, future prospects of biomarker development and other discovery approaches, including therapeutics.

Non-coding RNAs as emerging molecular targets of gallbladder cancer.

Gallbladder cancer is one of the most common cancers of biliary tract with aggressive pathophysiology, now emerging as a global health issue. Although minority of gallbladder cancer patients could receive such curative resection due to late diagnosis, this increases the survival rate. Lack of potential target molecule (s) for early diagnosis, better prognosis and effective therapy of gallbladder cancer has triggered investigators to look for novel technological or high throughput approaches to identify potential biomarker for gallbladder cancer. Intervention of non-coding RNAs in gallbladder cancer has been revealed recently. Non-coding RNAs are now widely implicated in cancer. Recent reports have revealed association of non-coding RNAs (microRNAs or miRNAs and long non-coding RNAs or lncRNAs) with gallbladder cancer. Here, we present an updated overview on the biogenesis, mechanism of action, role of non-coding RNAs, the identified cellular functions in gallbladder tumorigenesis, their prognostic & therapeutic potentials (efficacies) and future significance in developing effective biomarker(s), in future, for gallbladder.